Mechanism underlying cofactor-dependent proteolysis of von Willebrand Factor

项目总结 血管性血友病因子(von Willebrand factor，VWF)是一种大的多聚体血浆蛋白，在止血中起重要作用。VWF 被合成和分泌为含有25-50个启动子的超大(UL)多聚体。如果不是 ULVWF被血浆金属蛋白酶ADAMTS13处理后，可以启动生命的形成- 威胁血栓形成，如血栓性血小板减少性紫癜(TTP)。蛋白水解性裂解是如何 ADAMTS13对ULVWF的调节作用在生理条件下还不完全清楚。这个 解理位埋在VWF A2结构域的中心β-Sheet下面，拉力为 需要暴露裂解部位才能发生酶裂解。我们的初步研究已经 证明凝血因子VIII(FVIII)可能作为辅助因子促进切割 机械剪切作用下ADAMTS13对VWF的影响。利用我们独特的组合 两个实验室都有分子、生化和单分子生物物理工具，我们将测试 FVIII与VWF-D‘D3和其他邻近结构域如A2的结合假设 结构域可能导致中心A2的构象变化，从而暴露裂解位点 (Y1605-M1606)在机械力作用下更容易接触ADAMTS13。在目标1中，我们将确定 在没有和没有其他相邻结构域的情况下，A2的机械展开轮廓 在目标2中，我们将通过以下方式阐明A2和FVIII相互作用的分子机制 研究它们的变种和突变体；在目标3中，我们将确定 依赖FVIII的VWF在外力、动物模型和人中的蛋白水解性切割 健康的三聚氰胺。拟议项目的完成将有助于理解分子间的相互作用 在生理条件下的底物、酶和蛋白质辅因子之间，提供 开发预防和治疗TTP和其他疾病的新疗法的基本原理 血栓和炎症性疾病。

PROJECT SUMMARY von Willebrand factor (VWF), a large multimeric plasma protein, plays a critical role in hemostasis. VWF is synthesized and secreted as ultra-large (UL) multimers that contain 25-50 protomers. If not processed by a plasma metalloprotease ADAMTS13, ULVWF can initiate the formation of life- threatening thrombosis as in thrombotic thrombocytopenic purpura (TTP). How the proteolytic cleavage of ULVWF by ADAMTS13 is regulated under physiological conditions is not fully understood. The cleavage site is buried under the central β-sheet within the A2 domain of VWF, and tensile force is required to expose the cleavage site for enzymatic cleavage to occur. Our preliminary studies have demonstrated that coagulation factor VIII (FVIII) may function as a cofactor that facilitates the cleavage of VWF by ADAMTS13 under mechanic shear. Taking advantage of our unique combination of molecular, biochemical and single-molecule biophysical tools available in both laboratories, we will test the hypothesis that the binding of FVIII to VWF-D’D3 and other adjacent domains such as the A2 domain may result in conformational changes in the central A2, thus exposing the cleavage site (Y1605-M1606) more readily to ADAMTS13 under mechanical force. In Aim 1, we will determine the mechanical unfolding profile of A2 with or without other adjacent domains in the absence and presence of FVIII; in Aim 2, we will elucidate the molecular mechanism of A2 and FVIII interactions by investigating their variants and mutants; and in Aim 3, we will determine the physiological relevance of the FVIII-dependent proteolytic cleavage of VWF under force and in animal models and human with heareditary TTP. The completion of the proposed project will help understand the molecular interactions among substrate, enzyme, and protein cofactor under physiological conditions, which provides rationales for the development of novel therapeutics for the prevention and treatment of TTP and other thrombotic and inflammatory disorders.